The present invention relates to a stable DNA construct into which a target nucleic acid strand is incorporated for subsequent expression in a host. The construct includes inverted tandem repeats which form the stem of a stem-loop structure and which include one or more functional genetic elements having their function directed in cis-oriented fashion towards the single stranded target sequence.
Several researchers have created multi-functional, single stranded cloning vectors and cloning vectors which impart both single and double stranded DNA life cycle intermediates (i.e. phagmids). Additionally, naturally occurring stem-loop DNA and RNA structures are seen during the replicative cycle of viruses (predominantly) and cells and may have functional roles in DNA/RNA signaling mechanisms. Stem-loop structures have also been artificially produced primarily for DNA and RNA stability studies. However, so far as is known, there are no prior reports of the incorporation of genetic design into cloning vectors which allows for the stable formation of stem-loop structures (after conversion to single stranded intermediates) to perform useful functions to a target sequence(s) of interest that is located in the loop of the stem-loop structure by an isolated, functional, cis-acting double stranded genetic element (the stem).
The ability to form a stem-loop structure, after life cycle mediated single stranded conversion wherein the target sequence of choice (e.g., a cloned DNA fragment) is isolated as the so-called "loop" and is flanked by "functional" double stranded genetic elements (which form the so-called "stem") which direct the activities in cis-oriented fashion offers many advantages in performing useful manipulations to these target sequences. This structure, for instance, enables production of single stranded DNA probes which do not include the DNA sequences of the vector, thereby increasing their specificity over those made by known methods. Similarly, retroviral intermediates are constructed for drug delivery by triplex forming oligonucleotide production in vivo.